Crystallization of USP5 Zf-UBD #2

In my last post, I analyzed a structure of USP5 zinc-finger ubiquitin binding domain with a cysteine-glutathione disulphide bond. These crystals were not suitable for ligand soaking due to the formation of a disulphide bond at Cys195 with a glutathione additive that occupies the ubiquitin binding pocket in the adjacent molecule in the crystal lattice. Read More …

Structural Analysis of USP5 Zf-UBD

In a previous post, I determined a crystallization condition that produced nice 3D crystals of USP5 Zf-UBD that diffracted well. Rachel Harding, a postdoc at the SGC collected data images for these crystals and solved the structure using computational techniques. You can see the solved crystal structure and a more detailed analysis of the structure Read More …

USP5 Zf-UBD FP Assay Optimization

In my continued quest to develop biophysical assays to screen compounds against USP5 Zf-UBD, I have been working on optimizing fluorescence polarization (FP) assay conditions for a peptide displacement assay. The aim was to optimize buffer conditions to decrease the amount of protein required for the assay, and to determine the affinity of a ubiquitin Read More …

USP5 Zf-UBD Differential Scanning Fluorimetry Assay Development

It’s important to have more than one assay in place to validate experimental results. In addition to the fluorescence polarization assay, I am also trying to develop a differential scanning fluorimetry (DSF) assay. DSF measures the interaction of small molecules to the protein as the protein progressively unfolds. The melting temperature of USP5 Zf-UBD is Read More …

Analyzing the structural diversity of the USP5 Zf-UBD binding pocket

USP5 is one of many USP proteins that contains a zinc finger ubiquitin binding domain (Zf-UBD). My goal is to develop small molecules that inhibit USP5 selectively. So, it’s important to make sure that the pocket where the compound will bind is sufficiently and structurally different from other USPs. I did a multiple sequence alignment Read More …